Natural killer cells (NK cells) are human innate immune cells, of which the major representative surface markers are CD3−, CD56+, CD16+, CD57+, and CD8+. Such cells are characterized in that they contain perforin and telomerase, and can lyse the tumor cells sensitive to NK cells without prior sensitization, thereby killing the tumor cells. Natural killer cells are the major cells that mediate antibody-dependent cytotoxic effect, which express several ligands of tumor necrosis factor family and may result in apoptosis of a variety of target tumor cells. This mechanism of killing tumor cells through tumor cell apoptosis is more promising than the secretory, particles-mediated killing. Since most receptors on tumor cells are sensitive to death caused by apoptosis, natural killer cells are of great significance in terms of anti-tumor. The anti-tumor effect of natural killer cells is a fact to which the international mainstream medicine has no disputes.
Natural killer cell can cause apoptosis of various tumor cells and represents a kind of tumor immunotherapy, which has become the consensus of the international medical community. Natural killer cell is a type of human lymphocyte and is in small amounts, only accounting for 1-1.5% of the total lymphocytes. Therefore, how to obtain natural killer cells becomes the focus of the medical community.
Scientists cultured autologous peripheral blood cells from patients with cell inducing factors for 2-4 weeks and made CD3+ and CD56+ cells increase by 5-10%. The killer lymphocytes obtained therefrom were referred to as cytokine-induced killer (CIK) cells. Some scientists used the pre-prepared mesenchymal stem cells (MSC) to induce and cultivate natural killer cells in vitro, or used hematopoietic stem cells to induce and cultivate natural killer cells. All these methods for obtaining natural killer cells have certain limitations. For patients with advanced tumor, whether they will be treated with CIK cells depends on not only the decision of the physician, but also culture time and culture conditions. Natural killer cells require longer culture time than the disease progression in cancer patients, and thus have limited clinical application.
Natural killer lymphocytes are the medical community recognized cellular therapy for anti-tumor cell and immunity. Clinically, such cellular therapy comprises: isolating mononuclear cells from the peripheral blood from patients, adding cell inducing factors, performing culture and expansion in vitro for 2-6 weeks with the aim of allowing the mononuclear cells to develop into T lymphocytes (surface markers are CD3+ and CD56+), and returning the cells to the patients after they reach to a certain number. In normal human body, CD3+ and CD56+ cells are present in small number, while after expanded for 2-6 weeks through induced culture in vitro, CD3+ and CD56+ cells respectively reach at least 20% and 5%. Natural killer cell therapy gets some clinical efficacy in personalized treatment and significantly increases the five-year survival rate. However, there are still limitations in the development and promotion of clinical therapy.
Firstly, in this technique, the killer lymphocytes are cultured and expanded for clinical therapy by using mononuclear cells as carrier and adding cell inducing factors. So far, nearly all the NK cell therapies worldwide adopt one-to-one personalized treatment. That is, mononuclear cells from a patient are directly collected or indirectly collected by isolating mononuclear cells from the donor blood of a patient, and then the mononuclear cells were induced, expanded in vitro, and used to treat various solid tumors in the patient. Since this traditional therapy is a one-to-one personalized treatment method, it can only be carried out at bedside in hospital.
Secondly, in this therapy, it generally takes 2-6 weeks to prepare natural killer cells. For patients with advanced tumor, especially the patients subjected to radiotherapy and chemotherapy after surgery, some of them may pass away before the preparation of cells is completed, or the resultant cells are returned.
Thirdly, as for the one-to-one therapy, a complete processes of cell induction and expansion culture need to be conducted for each person. From the view of laboratory management, it is hard to control internal quality, not to mention the external quality. Although this is an excellent medical technology, it is difficult to promote and spread. Not all patients share equal therapeutic opportunity.
Fourthly, theoretically, CIK cells prepared as described above are induced, cultured and expanded from adult stem cells, which are contained in the culture medium in large amount and can produce CIK cells through unlimited expansion. However, clinically, after expanded for 5-6 weeks, their expansion rate decreases significantly and the expansion amount (doubling number) is obviously less than that at the early stages. Furthermore, with the expansion generation increased, their immune killing effects on tumor cells also reduce. Therefore, it is vital to control and grasp the number of subculture of natural killer cells, while maintaining the biological activity of natural killer cells is more important.
Fifthly, in the conventional methods, mononuclear cells or hematopoietic stem cells derived from patients are used as primary cells, and cytokines are used to induce CIK cells. As for these mononuclear cells from tumor patients, the mononuclear cells of some patients share common antigenicity with their tumor cells; therefore, the induced anti-tumor ability of CIK cells tends to be poor when the mononuclear cells from these patients are used as primary cells.
It has been verified by scientists that allogeneic umbilical cord blood karyocytes contain abundant stem cells, the biological activity of such stem cells is superior to that of the stem cells derived from autologous bone marrow and autologous peripheral blood. This is a population of stem cells with the potential of multiple differentiation and expansion generation. Stem cells are have many types, comprising hematopoietic stem cells (HSC), mesenchymal stem cells (MSC), unrestricted somatic stem cells (USSCs), cord blood-derived embryonic-like stem cells (CBEs), cord blood-derived multipotent adult progenitor cells (CB-MPCs).
Although the allogeneic peripheral blood karyocytes contain less abundant stem cells than umbilical cord blood, they contain hematopoietic stem cells, mesenchymal stem cells, small amounts of cord blood-derived multipotent adult progenitor cells (CB-MPCs).
There are numerous methods for extracting and separating karyocytes from umbilical cord blood and peripheral blood. The extraction methods involved in the present invention are the method in the applicant's prior granted patent “A kit for separating karyocytes in vitro and application method thereof” (Patent No. ZL200610106875.5) and the technique for using FICOLL® or PERCOLL® density gradient media (GE Healthcare Bioprocess R&D AB, Uppsala, Sweden) centrifugation to isolate and screen out karyocytes.